Cytochrome c peroxidase (CcP) is a detoxification enzyme, designed to maintain low levels of intracellular H2O2 levels by reducing the peroxide to water. The catalytic mechanism involves reduction of H2O2 with associated oxidation of CcP to an intermediate called CcP compound I (CcPI). CcPI contains an oxidized tryptophan radical as well as an Fe(IV) site. CcPI is reduced back to the native state by ferrocytochrome c. Reduction of CcPI by electron transfer from cytochrome c is the focus of this research proposal. The cytochrome c/CcP system provides an exceptional opportunity to explore details of long-range electron transfer within dynamic, electrostatically-stabilized protein-protein complexes. Structural aspects of the system are very well characterized with crystal structures available for both CcP and cytochrome c. Crystal structures for 1:1 complexes of yeast iso-1 cytochrome c/CcP and horse cytochrome c/CcP are also available. In spite of the exceptional structural information available for the system, the kinetic and mechanical aspects of the electron transfer processes in the cytochrome c/CcP system are puzzling. In addition to a high-affinity 1:1 complex of cytochrome c and CcP, recent studies provide definitive evidence for a second, low-affinity binding site on CcP, leading to formation of 2:1 complexes. Most of the controversy surrounding the cytochrome c/CcP system concerns the role of cytochrome c bound to the low affinity binding site. Two fundamentally different mechanisms have been proposed for electron transfer between cytochrome c and CcP. One mechanism postulates that electron transfer only occurs via a single high-affinity binding site while the second postulates that cytochrome c can transfer electrons to the heme site in CcP via both binding domains, with the low-affinity domain having the higher electron transfer activity. The primary goal of this proposal is to determine which of these two mechanisms is correct by using specific covalent complexes of CcP and cytochrome c to explore the electron transfer activity of cytochrome c bound to the high- and low-affinity sites on CcP. The covalent complexes will be crosslinked through disulfide bonds using modified CcP and cytochrome c molecules in which cysteine residues are engineered into specific locations on the two proteins.